The present invention concerns a method for the detection of 5-methylcytosine in DNA. 5-Methylcytosine is the most frequent covalently modified base in the DNA of eukaryotic cells. It plays an important biological role, among other things, in the regulation of transcription, in genetic imprinting and in tumorigenesis (for review: Millar et al.: Five not four: History and significance of the fifth base. In: The Epigenome, S. Beck and A. Olek (eds.), Wiley-VCH Publishers, Weinheim 2003, pp. 3-20). The identification of 5-methylcytosine as a component of genetic information is thus of considerable interest. The detection of methylation is difficult, since cytosine and 5-methylcytosine have the same base pairing behavior. Many of the conventional detection methods based on hybridization thus cannot distinguish between cytosine and methylcytosine. In addition, the methylation information is completely lost in a PCR amplification.
The conventional methods for methylation analysis operate essentially according to two different principles. In the first, methylation-specific restriction enzymes are used, and in the second, a selective chemical conversion of unmethylated cytosines to uracil occurs (e.g. by means of bisulfite treatment, see, e.g.: DE 101 54 317 A1; DE 100 29 915 A1). The DNA that has been pretreated enzymatically or chemically is then amplified and can be analyzed in different ways (for review: WO 02/072880 pp. 1 ff). Methods which can detect methylation in a sensitive and quantitative manner are of great interest. This is true due to the important role of cytosine methylation in the development of cancer, particularly with respect to diagnostic applications. Of particular importance are methods which permit detection of deviant methylation patterns in body fluids, e.g., in serum. Unlike unstable RNA, DNA is often encountered in body fluids. The DNA concentration in blood is in fact increased in destructive pathological processes such as cancer disorders. The diagnosis of cancer by means of a methylation analysis of tumor DNA found in body fluids is thus possible and has in fact been described many times (see e.g.: Palmisano et al.: Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res. 2000 Nov. 1; 60(21):5954-8). A particular problem however, consists of the fact that in body fluids, in addition to the DNA with the methylation pattern typical of disease there is also a large quantity of DNA of identical sequence but of another methylation pattern to be found. The diagnostic methods must thus be able to detect small quantities of particularly methylated DNA against an intense background of DNA of the same sequence but of another methylation pattern (hereinafter also referred to as ‘background DNA’).
The conventional methods for methylation analysis solve this problem only to a limited extent. Usually the chemically pretreated DNA is amplified by means of a PCR method. A selective amplification of only methylated (or in the opposite approach, of only unmethylated) DNA is assured by the use of methylation-specific primers or blockers. The use of methylation-specific primers is known as a “methylation-sensitive PCR” (“MSP”; Herman et al.: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996 Sep. 3; 93(18):9821-6). A comparably sensitive method is the so-called “HeavyMethyl” method. Here, a specific amplification of only the originally methylated (or unmethylated) DNA is obtained by use of methylation-specific blocker oligomers (for review: WO 02/072880). Both MSP and the “HeavyMethyl” method can be applied as quantifiable real-time variants. These make possible the detection of the methylation status of a few positions directly in the course of the PCR without the need for a subsequent analysis of the products (“MethyLight”—WO 00/70090; U.S. Pat. No. 6,331,393). One such embodiment is the “Taqman” method. This technique uses probe molecules which bear a fluorescent-dye/quencher pair. The probes hybridize in a sequence-specific manner to the amplificates and are decomposed in the course of the next amplification cycle by the exonuclease activity of the polymerase. A detectable fluorescent signal arises due to the separation of quencher and dye (see, e.g., Eads et al.: MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 2000 Apr. 15; 28(8):E32).
Another MethyLight embodiment is the so-called Lightcycler method. In this case, two different probes are utilized, which hybridize to the amplificate in the direct vicinity of one another, and then produce a detectable signal via fluorescence-resonance energy transfer (FRET).
The applicability of this method for a sensitive and specific detection of methylated DNA against a large background of unmethylated DNA is of course limited. The danger exists that false-positive results will occur by means of a nonspecific amplification of background DNA. In order to increase the specificity of the amplification, it is thus necessary to use primers or blocker sequences, in which several methylation-specific positions are contained. These sequence requirements, in turn, limit the applicability of the method.
Based on the particular biological and medical importance of cytosine methylation and due to the above-mentioned disadvantages of the prior art, there is a great technical need for the development of high-performance methods for methylation analysis. Such a method is described in the following.
According to the invention, the DNA to be investigated is first chemically pretreated, after which it is hybridized to oligonucleotide probes and then reacted with DNA repair enzymes. One embodiment of the invention permits a specific decomposition of background DNA. A selective amplification of only the DNA whose methylation status will be detected, is thereby facilitated. A very sensitive and very specific analysis of methylation is thus enabled. The field of application of the method according to the invention is thus broader than that of the methodologies already known (see above). Methylation-specific primer or blocker sequences are not necessary to the same extent.
Another embodiment of the method according to the invention utilizes hybridization and the application of DNA repair enzymes, not for the decomposition of background DNA, but directly for the detection of the methylation status. A similar method for mutation analysis is described under the name “Midas” (Bazar et al.: Mutation identification DNA analysis system (MIDAS) for detection of known mutations. Electrophoresis. 1999 June; 20(6):1141-8; U.S. Pat. No. 5,656,430). In this case, an oligonucleotide is hybridized to the DNA to be investigated. An erroneous base pairing is formed at the mutation to be detected, which is then recognized by a mismatch repair enzyme. The probe is cleaved at this site, and the fragments can be detected by different methods. If an excess of probe is used, then the process can be reiterated. Another similar method for mutation analysis has been described by Zhang et al. (An amplification and ligation-based method to scan for unknown mutations in DNA. Hum Mutat. 2002 August; 20(2):139-47). Here, the DNA to be investigated is first amplified by means of a PCR. Then the amplificates are cross-hybridized with the formation of erroneous base pairings. After this, breaks in the single strand are introduced by reaction with repair enzymes. Finally, primers are ligated specifically to the cleaved DNA, by means of which the mutation can be specifically detected.
The application of these two methods to methylation analysis is described for the first time below. Based on the particular biological and medical importance of cytosine methylation and due to the disadvantages of the known methods, the discovery of this advantageous new technology represents an important technical advance.
All patents, patent applications and publications referred to or cited herein, or from which a claim for benefit of priority has been made, are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification.